The extracellular matrix in vertebrates provides structural support to the cell, aids in osmo-regulation, and is particularly important in mediating cell-cell interactions in soft connective tissues, such as cartilage and skin. A major component of the extracellular matrix is hyaluronan (HA), which is an extracellular linear polysaccharide containing alternating N-acetylglucosamine (NAG) and glucuronic acid (GA) residues. HA affects many physiological processes, from cell adhesion and migration to cell differentiation and embryological development. Because of its broad impact on human physiology, a large number of pathological conditions, including many forms of cancer, autoimmune diseases, inflammatory processes, and rheumatoid arthritis, correlate with altered expression levels of HA. On a molecular level, HA is produced inside the cell by the membrane-embedded hyaluronan synthase (HAS). HAS is a remarkable enzyme. It not only catalyzes the synthesis of HA from UDP-activated substrates, but it also transports the growing polymer across the cell membrane to deposit it within the extracellular matrix. In order to accomplish this task, HAS has to fulfill several functions. The enzyme binds the substrates UDP-NAG and -GA, it catalyzes the glycosyl transfer reaction to form HA, and it translocates the growing polymer across the cell membrane through a pore formed by its own transmembrane region. To understand how HA exerts its physiological function and to produce HA polymers with defined properties for biomedical applications, we must first unravel how HAS synthesizes HA and how it deposits the polymer in the extracellular matrix. To this end, we propose three aims that will reveal the assembly of biologically active HAS subunits in native lipid membranes, will identify the interactions between HAS and the translocating HA polymer, and will allow us to determine the structure of HAS by X-ray crystallography. First, we will combine co-immunoprecipitation studies with chemical cross-linking and photobleaching techniques to visualize HAS oligomers in native membranes. The low-resolution structural data will then be integrated with high-resolution structures of monomeric HAS to reconstruct the native, membrane-embedded HAS oligomer. Second, the interactions of HAS with the translocating HA polymer will be mapped by introducing UV-inducible cross-linkers into the TM-region of HAS. Cross-linking during HA translocation will identify positions that are in close proximity to the polysaccharide, thus delineating the physico-chemical properties of the HA transmembrane channel. Third, biochemical and low resolution structural data will be integrated with a high-resolution structure of HAS obtained by X-ray crystallography. Determining the structure of HAS both in a detergent-solubilized but also in a membrane-embedded state will reveal the architecture and oligomeric form of the synthase, allowing us to delineate the mechanism by which this marvelous enzyme synthesizes one of the most abundant extracellular polysaccharides in the human body.